The addition of peptide and protein fusion tags to recombinant proteins is widely employed in biochemical studies (Terpe, K. Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Appl. Microbiol. Biotechnol. 60, 523-533 (2003); Chelur, D., Unal, O., Scholtyssek, M. & Strickler, J. Fusion tags for protein expression and purification. in BioPharm Int. 2008 June supplement, Guide to Protein Production 38-46, Advanstar Communications, Inc., Woodland Hills, Calif., 2008). Fusion tags exist for a variety of applications, including affinity purification, solubility enhancement, and protein detection (Terpe, K. Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Appl. Microbiol. Biotechnol. 60, 523-533 (2003); Chelur, D., Unal, O., Scholtyssek, M. & Strickler, J. Fusion tags for protein expression and purification. in BioPharm Int. 2008 June supplement, Guide to Protein Production 38-46 (Advanstar Communications, Inc., Woodland Hills, Calif., 2008); Nilsson, J., Stahl, S., Lundeberg, J., Uhlen, M. & Nygren, P.A. Affinity fusion strategies for detection, purification, and immobilization of recombinant proteins. Protein Expr. Purif. 11, 1-16 (1997); Arnau, J., Lauritzen, C., Petersen, G.E. & Pedersen, J. Current strategies for the use of affinity tags and tag removal for the purification of recombinant proteins. Protein Expr. Purif. 48, 1-13 (2006); Lichty, J.J., Malecki, J.L., Agnew, H.D., Michelson-Horowitz, D.J. & Tan, S. Comparison of affinity tags for protein purification. Protein Expr. Purif. 41, 98-105 (2005)). Affinity purification methods are popular because they can dramatically lower purification time, often require minimal purification steps, and can be used to obtain greater than 90% purity with high yields (Terpe, K. Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Appl. Microbiol. Biotechnol. 60, 523-533 (2003); Nilsson, J., Stahl, S., Lundeberg, J., Uhlen, M. & Nygren, P.A. Affinity fusion strategies for detection, purification, and immobilization of recombinant proteins. Protein Expr. Purif. 11, 1-16 (1997); Lichty, J.J., Malecki, J.L., Agnew, H. D., Michelson-Horowitz, D.J. & Tan, S. Comparison of affinity tags for protein purification. Protein Expr. Purif. 41, 98-105 (2005)). However, affinity-tagged proteins are purified using conditions (buffer, additives, etc.) specific to the fusion tag, and not the protein of interest (Chelur, D., Unal, O., Scholtyssek, M. & Strickler, J. Fusion tags for protein expression and purification. in BioPharm Int. 2008 June supplement, Guide to Protein Production 38-46 (Advanstar Communications, Inc., Woodland Hills, Calif., 2008)). Thus, no single affinity procedure can be used to purify every protein, resulting in the need for a wide variety of fusion tags and the continuing development of novel affinity purification methods (Chelur, D., Unal, O., Scholtyssek, M. & Strickler, J. Fusion tags for protein expression and purification. in BioPharm Int. 2008 June supplement, Guide to Protein Production 38-46 (Advanstar Communications, Inc., Woodland Hills, Calif., 2008)). Recently, fusion tags detectable by the naked eye have been developed for continuous tracking of protein expression, production, and purification (Chelur, D., Unal, O., Scholtyssek, M. & Strickler, J. Fusion tags for protein expression and purification. in BioPharm Int. 2008 June supplement, Guide to Protein Production 38-46 (Advanstar Communications, Inc., Woodland Hills, Calif., 2008)). For instance, protein fusions based on the yellow human flavin mononucleotide (FMN)-binding domain and the red mosquito cytochrome b5 have been developed. In addition, the CHERRY™ Express (Delphi Genetics) kit for fusing an 11-kDa heme protein that also allows for protein quantitation is commercially available (Finn, R.D., Kapelioukh, L. & Paine, M.J.I. Rainbow tags: a visual tag system for recombinant protein expression and purification. Biotechniques 38, 387-392 (2005)). These colored tags aid visualization and quantitation, but for affinity purification, additional tags are needed.
Citation or identification of any reference in Section 2, or in any other section of this application, shall not be considered an admission that such reference is available as prior art to the present invention.